Name: Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade
SKU: ab185050
Rating: 5 (4 reviews)

Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) is a rabbit monoclonal antibody detecting EHMT2/G9A in Western Blot, Flow Cytometry (Intra), Flow Cytometry, IP, IHC-P, ICC/IF, ChIP. Suitable for Human, Mouse, Rat.

- KO validated for confirmed specificity

- Biophysical QC for unrivalled batch-batch consistency

- Over 30 publications

Images

Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (AB185050), expandable thumbnail

Publications

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

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Reactivity data

Select an application

Product promiseTestedExpectedPredictedNot recommended

	ChIC/CUT&RUN-seq	IP	WB	ICC/IF	ChIP-seq	Flow Cyt (Intra)	IHC-P
Human	Tested	Tested	Tested	Tested	Tested	Tested	Tested
Mouse	Expected	Expected	Tested	Expected	Expected	Expected	Tested
Rat	Expected	Expected	Tested	Expected	Expected	Expected	Tested

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 5 µg	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse	Dilution info 5 µg	Notes -
Species Rat	Dilution info 5 µg	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/50	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/1000	Notes -
Species Rat	Dilution info 1/1000	Notes -
Species Human	Dilution info 1/1000	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/1000	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 8 µg for 10⁷ Cells	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Human	Dilution info 1/150	Notes -

Expected
Expected

Species	Dilution info	Notes
Species Mouse, Rat	Dilution info Use at an assay dependent concentration.	Notes -

Tested
Tested

Species	Dilution info	Notes
Species Mouse	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Rat	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Species Human	Dilution info 1/500	Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

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Target data

See full target information EHMT2

Function

Histone methyltransferase that specifically mono- and dimethylates 'Lys-9' of histone H3 (H3K9me1 and H3K9me2, respectively) in euchromatin. H3K9me represents a specific tag for epigenetic transcriptional repression by recruiting HP1 proteins to methylated histones. Also mediates monomethylation of 'Lys-56' of histone H3 (H3K56me1) in G1 phase, leading to promote interaction between histone H3 and PCNA and regulating DNA replication. Also weakly methylates 'Lys-27' of histone H3 (H3K27me). Also required for DNA methylation, the histone methyltransferase activity is not required for DNA methylation, suggesting that these 2 activities function independently. Probably targeted to histone H3 by different DNA-binding proteins like E2F6, MGA, MAX and/or DP1. May also methylate histone H1. In addition to the histone methyltransferase activity, also methylates non-histone proteins: mediates dimethylation of 'Lys-373' of p53/TP53. Also methylates CDYL, WIZ, ACIN1, DNMT1, HDAC1, ERCC6, KLF12 and itself.

Alternative names

BAT8, C6orf30, G9A, KMT1C, NG36, EHMT2, Histone-lysine N-methyltransferase EHMT2, Euchromatic histone-lysine N-methyltransferase 2, HLA-B-associated transcript 8, Histone H3-K9 methyltransferase 3, Lysine N-methyltransferase 1C, Protein G9a, H3-K9-HMTase 3

Key facts

Isotype: IgG
Host species: Rabbit
Storage buffer: pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Form: Liquid
Clonality: Monoclonal
Immunogen: The exact immunogen used to generate this antibody is proprietary information.
Clone number: EPR18894
Purification technique: Affinity purification Protein A
Concentration

Storage

Shipped at conditions: Blue Ice
Appropriate short-term storage duration: 1-2 weeks
Appropriate short-term storage conditions: +4°C
Appropriate long-term storage conditions: -20°C
Aliquoting information: Upon delivery aliquot
Storage information: Avoid freeze / thaw cycle

Notes

What is this antibody validated in?

Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) is a rabbit recombinant monoclonal antibody and is validated for use in Western Blot (WB), Flow Cytometry (Intra), Flow Cytometry (Flow Cyt), Immunoprecipitation (IP), Immunohistochemistry (IHC-P), Immunocytochemistry/immunofluorescence (ICC/IF), ChIP in Human, Mouse, Rat samples.

What is the molecular weight of EHMT2/G9A?

Anti-EHMT2/G9A [EPR18894] - ChIP Grade (ab185050) specifically detects a band for EHMT2/G9A (UniProt: Q96KQ7) at a molecular weight of 132kDa.

Trusted by the scientific community

Anti-EHMT2/G9A [EPR18894] - ChIP Grade (ab185050) was first used in a scientific publication in 2015 and has been cited over 30 times in peer-reviewed journals.

Trial sizes available!

Test your antibody or perform pre-screening before committing to a larger quantity. Sold in 10µl. Discover our selection of trial-size antibodies.

Specificity confirmed

The specificity of Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) has been confirmed by Western blot testing in EHMT2 Knockout HAP1 cell line, ab265149.

Other related products

We have a range of other formats of antibody clone [EPR18894] also available for your convenience:
ab185050, Alexa Fluor® 488 - ab218359, Carrier free - ab240289

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

EHMT2 also known as G9a is a methyltransferase enzyme with a molecular mass of approximately 165 kDa. It modifies histones by adding methyl groups to lysine 9 on histone H3 which results in chromatin structure changes and influences gene expression. EHMT2/G9a expresses in various tissues including brain heart and liver showing its widespread role in different physiological processes. As a well-studied protein researchers often analyze EHMT2 activity using assays like ELISA and western blot.

Biological function summary

EHMT2/G9a regulates gene expression by altering chromatin accessibility. It is part of a chromatin remodeling complex interacting with proteins such as ANMTA02. Through its methyltransferase activity it maintains an epigenetic silence state important for embryonic development differentiation and cellular identity. EHMT2/G9a plays a significant role in the repression of transcriptional activity of tumor suppressor genes and developmental genes.

Pathways

G9a functions in epigenetic signaling pathways and influences processes like the cell cycle and DNA damage repair. It interacts with proteins in the Polycomb repressive complex such as EZH2 to regulate gene silencing. Additionally in the DNA damage response pathway G9a can associate with histone deacetylases to stabilize chromatin and prevent unwarranted gene expression.

Associated diseases and disorders

Aberrant regulation of EHMT2/G9a associates with cancer and neurodevelopmental disorders. In various cancers such as leukemia G9a suppresses tumor suppressor genes aiding tumor progression. Connections between G9a and proteins like DNMT1 highlight its role in maintaining a hypermethylation state in cancerous cells. In neurodevelopmental disorders altered expression of G9a affects synaptic plasticity linking it with cognitive impairments and autism spectrum disorders.

Product promise

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17 product images

Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Lanes 1-4: Merged signal (red and green). Green - ab185050 observed at 160 kDa. Red - loading control Anti-GAPDH antibody [6C5] - Loading Control ab8245 observed at 37 kDa.
ab185050 Anti-EHMT2/G9A antibody [EPR18894] was shown to specifically react with EHMT2/G9A in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human EHMT2 (G9A) knockout HeLa cell line ab265149 (knockout cell lysate Human EHMT2 (G9A) knockout HeLa cell lysate ab257080) was used. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab185050 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) at 1/1000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: EHMT2/G9A knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human EHMT2 (G9A) knockout HeLa cell line (Human EHMT2 (G9A) knockout HeLa cell line ab265149)
Lane 3: HEK-293 cell lysate at 20 µg
Lane 4: HepG2 cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/20000 dilution
Performed under reducing conditions.
Predicted band size: 132 kDa
Observed band size: 160 kDa
Immunocytochemistry/ Immunofluorescence - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling EHMT2/G9A with ab185050 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green).
Confocal image showing nuclear staining on HeLa cell line.
The nuclear counterstain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:-
-ve control 1: ab185050 at 1/1000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.
ChIP-sequencing - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 8 μg of ab185050 [EPR18894]. ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Nucleus staining on rat kidney is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Lanes 1 - 4: Merged signal (red and green). Green - ab185050 observed at 160 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
ab185050 was shown to recognize EHMT2/G9A when EHMT2/G9A knockout samples were used, along with additional cross-reactive bands. Wild-type and EHMT2/G9A knockout samples were subjected to SDS-PAGE. ab185050 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) at 1/1000 dilution
Lane 1: Wild-type HAP1 cell lysate at 20 µg
Lane 2: EHMT2/G9A knockout HAP1 cell lysate at 20 µg
Lane 3: HEK293 cell lysate at 20 µg
Lane 4: HepG2 cell lysate at 20 µg
Predicted band size: 132 kDa
Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Blocking/Dilution buffer: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).
All lanes: Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) at 1/1000 dilution
Lane 1: HEK-293 (Human epithelial cells from embryonic kidney) whole cell lysate at 20 µg
Lane 2: Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg
Lane 3: HepG2 (Human liver hepatocellular carcinoma) whole cell lysate at 20 µg
Lane 4: NCCIT (Human pluripotent embryonic carcinoma) whole cell lysate at 20 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 132 kDa
Observed band size: 160 kDa, 170 kDa
Exposure time: 30s
Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Blocking/Dilution buffer: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).
All lanes: Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) at 1/1000 dilution
All lanes: Human fetal heart lysate at 10 µg
Secondary
All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution
Predicted band size: 132 kDa
Observed band size: 160 kDa, 170 kDa
Exposure time: 3min
Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Blocking/Dilution buffer: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).
All lanes: Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) at 1/1000 dilution
All lanes: Human fetal kidney lysate at 10 µg
Secondary
All lanes: Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution
Predicted band size: 132 kDa
Observed band size: 160 kDa, 170 kDa
Exposure time: 8s
Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Blocking/Dilution buffer: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).
All lanes: Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) at 1/1000 dilution
Lane 1: F9 (Mouse embyro testicular cancer cell line) whole cell lysate at 10 µg
Lane 2: Neuro-2a (Mouse neuroblastoma cells) whole cell lysate at 10 µg
Lane 3: LLC1 (Mouse lung carcinoma cell line) whole cell lysate at 10 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 132 kDa
Observed band size: 160 kDa, 170 kDa
Exposure time: 5s
Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Blocking/Dilution buffer: 5% NFDM/TBST.
The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID 16702210).
All lanes: Western blot - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050) at 1/1000 dilution
Lane 1: C6 (Rat glial tumor cells) whole cell lysate at 10 µg
Lane 2: RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg
Lane 3: PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg
Lane 4: NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate at 10 µg
Secondary
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution
Predicted band size: 132 kDa
Observed band size: 160 kDa, 170 kDa
Exposure time: 8s
Immunoprecipitation - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
EHMT2/G9A was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate with ab185050 at 1/50 dilution.
Western blot was performed from the immunoprecipitate using ab185050 at 1/1000 dilution.
VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used for detection at 1/10000 dilution.
Lane 1: HeLa whole cell lysate 10ug (Input).
Lane 2: ab185050 IP in HeLa whole cell lysate.
Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab185050 in HeLa whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 30 seconds.
All lanes: Immunoprecipitation - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Predicted band size: 132 kDa
Observed band size: 160 kDa, 170 kDa
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Nucleus staining on epithelial cells of the normal Human colon is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Immunohistochemical analysis of paraffin-embedded Human gastric adenocarcinoma tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Nucleus staining on tumor cells of the gastric adenocarcinoma is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling EHMT2/G9A with ab185050 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Nucleus staining on hepatocytes of the mouse liver is observed.
Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/500 dilution.
Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Immunocytochemistry/ Immunofluorescence - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling EHMT2/G9A with ab185050 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green).
Confocal image showing no staining on MCF7 cell line, as MCF7 cells have a very low level expression of EHMT2/G9A.
The nuclear counterstain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:-
-ve control 1: ab185050 at 1/70 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.
Flow Cytometry (Intracellular) - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling EHMT2/G9Awith ab185050 at 1/150 dilution (red) compared with a rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.
ChIC/CUT&RUN sequencing - Anti-EHMT2/G9A antibody [EPR18894] - ChIP Grade (ab185050)
ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL. 2.5X10^5 of Human wild-type HeLa cell line (ab255928) or EHMT2 (G9A) knockout HeLa cell line (Human EHMT2 (G9A) knockout HeLa cell line ab265149) were used along with 5µg of ab185050 [EPR18894]. Assay Quality Control was conducted using 5µg Anti-CTCF (Anti-CTCF antibody [EPR18253] - ChIP Grade ab188408) on the same cell lines. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.? The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.

Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.